The human immune system is equipped with several defense mechanisms to respond to bacterial, viral, or parasitic infection and injury. One of such defense mechanisms is the complement system, which plays a role both in innate and acquired immunity (see e.g. Cooper (1985), Adv. Immunol. 61:201-283; Liszewski et al. (1996), Adv. In Immunol. 61:201-282; Matsushita (1996), Microbiol. Immunol. 40:887-893; Sengelov (1995), Critical Review in Immunol. 15:107-131). The complement system directly and indirectly contributes to both innate inflammatory reactions as well as cellular (i.e. adaptive) immune responses. This array of effector functions is due to the activity of a number of complement components and their receptors on various cells. One of the principal functions of complement is to serve as a primitive self-nonself discriminatory defense system. This is accomplished by coating a foreign material with complement fragments and recruiting phagocytic cells that attempt to destroy and digest the “intruder”.
Complement refers to a group of plasma proteins that are known to be necessary for antibody-mediated bactericidal activity. The complement system is composed of more than 30 distinct plasma and membrane bound proteins involving three separate pathways: classical, alternative and the lectin pathway. The C3 protein sits at the juncture of the classical and alternative pathways and represents one of the critical control points. Cleavage of C3 yields C3a and C3b. C3b molecules then react with a site on the C4b protein, creating a C3b-C4b•C2b complex that acts as a C5 convertase. Proteolytic activation of C5 occurs only after it is bound to the C3b portion of the C5 convertase on the surface of an activator (e.g., the immune complex). Like C3, C5 is also cleaved by C2b to produce fragments designated C5a (16,000 Da) and C5b (170,000 Da). The C5b molecule combines with the proteins of the terminal components to form the membrane attack complex described below. C5a is a potent inflammatory mediator and is responsible for many of the adverse reactions normally attributed to complement activation in various clinical settings.
The classical pathway (CP) of complement activation is activated primarily by immune complexes (ICs), but also by other proteins such as C-Reactive Protein, Serum Amyloid Protein, amyloid fibrils, and apoptotic bodies (Cooper, 1985).
The lectin pathway, discovered in the 1990s (Matsushita, 1996) is composed of lectins like mannose binding protein (or mannan binding lectin, MBL) and two MBL-associated serine proteases (MASP-1 and MASP-2) (see Wong et al, 1999). Upon activation of MBL•MASP-1•MASP-2, the MASP protease components cleave C4 and C2 forming a CP C3 convertase described above.
In the alternative pathway (AP) of complement activation, C3 is cleaved to form C3b in a mostly hydrolyzed and inactivated form. This process has been termed “C3 tickover,” a continuous and spontaneous process that ensures that whenever an activating surface (a bacterium, biomaterial, etc) presents itself, reactive C3b molecules will be available to mark the surface as foreign. Eventually, a C3b molecule attaches to one of the C3 convertase sites by direct attachment to the C3b protein component of the enzyme. This C3b-C3b•Bb complex is the alternative pathway C5 convertase and, in a manner reminiscent of the CP C5 convertase, converts C5 to C5b and C5a.
All three pathways lead to a common point: cleavage of C5 to produce C5b and C5a. C5a is a potent inflammatory mediator. The production of C5b initiates the formation of a macromolecular complex of proteins called the membrane attack complex (MAC) that disrupts the cellular lipid bilayer, leading to cell death. Even at sublytic levels, formation of MAC on host cells results in a number of activation responses (elevated Ca+2, arachadonic acid metabolism, cytokine production).
Various types of control mechanisms have evolved to regulate the activity of the complement system at numerous points in the cascade (Liszewski et al, 1996). These mechanisms and include: 1) decay (dissociation) of converatase complexes, 2) proteolytic degradation of active components that is facilitated by several cofactors, 3) protease inhibitors, and 4) association of control proteins with terminal components that interfere with MAC formation. Without these important control elements, unregulated activation of the cascade results in overt inflammatory damage to various tissues and has been demonstrated to contribute to the pathology of many diseases.
Except for the cytotoxic action of the MAC, most of the biological responses elicited by complement proteins result from ligand-receptor-mediated cellular activation (Sengelov, 1995). The ability of complement to function in the opsonization of foreign elements is accomplished in large part by a set of receptors that recognize various C3 and C4 fragments bound to these foreign surfaces. These proteins help mediate the cell-cell interactions necessary for such activities as chemotaxis and cytotoxic killing.
In contrast to the above-discussed ligands, which remain attached to activating surfaces, C3a, C4a, and C5a are small cationic polypeptides that diffuse into the surrounding medium to activate specific cells. These peptides are called anaphylatoxins because they stimulate histamine release from mast cells and cause smooth muscle contraction, which can produce increased vascular permeability and lead to anaphylactic shock. These activities are lost when the peptides are converted to their des arg analogs (i.e., with the loss of their carboxyl terminal arginine residue). This occurs rapidly in vivo and is catalyzed by serum carboxypeptidase N.
In addition to its anaphylatoxic properties, C5a and C5a-desarg bind to specific receptors originally found on neutrophils and monocytes. Recently the receptors for both C5a and C3a have been cloned and sequenced. The C5aR (CD88) has been shown to be expressed on endothelial cells (EC), hepatocytes, epithelial cells (lung and kidney tubules), T cells, cells in the CNS as well as on the myeloid cell lines. In addition, expression levels of C5aRs are increased on EC and hepatocytes by exposure to LPS and IL-6. In myeloid cells (neutrophils and monocytes), the C5a-receptor interaction leads to a variety of responses, including chemotaxis of these cells into an inflammatory locus; activation of the cells to release the contents of several types of secretory vesicles and produce reactive oxygen species that mediate cell killing; increased expression of CR1, CR3, and LFA-1, resulting in cellular hyperadherence; and the production of other mediators such as various arachidonic acid metabolites and cytokines, e.g., IL-1, -6, and -8. Many of the adverse reactions seen during extracorporeal therapies, such as hemodialysis, are directly attributable to C5a production. C3aRs are expressed on a variety of cell types including eosinophils, neutrophils, monocytes, mast cells, astrocytes (in the CNS), as well as γ-IFN-activated T cells. In eosinophils, C3a elicits responses similar to C5a, including intracellular calcium elevation, increases endothelial cell adhesion, and the generation of reactive oxygen intermediates.
While complement is beneficial for fighting against pathogens, inappropriate or excessive activation of the complement system can lead to inadvertent tissue damage and cytotoxic responses. In order to control this, nature has built into the complement system several control mechanisms utilizing both plasma and cell surface proteins to limit the amount of activation and prevent damage to host tissues. However, in several disease settings complement activation is not adequately controlled and results in tissue damage.
Over activation of the complement system has been shown to play a role in a wide range of diseases including autoimmune diseases such as: glomerulonephritis (McLean (1993) Pediatr. Nephrol. 7:226; Couser et al. (1985) Kidney Inst. 29:879; Nangaku et al. (2002) J. Am. Soc. Nephrol. 13:928-936; Sato et al. (1999) J. Am. Soc. Nephrol. 10:1242-1252); rheumatoid arthritis (Mollnes et al. (1986) Arthritis Rheum. 29:715; Jones et al. (1994) Br. J. Rheum. 33:707; Fava et al. (1993) Clin. Exp. Immunol. 94:261; Strachan et al. (2001) British J. Pharm. 134:1778-1786); type II collagen-induced arthritis (Watson & Townes (1985) J. Exp. Med. 162:1878); psoriasis (Strachan et al. 2001), systemic lupus erythamatosis (Wang et al. (1996) Proc. Natl. Acad. Sci. 93:8563; Stracha et al. 2001); transplantation rejection (Wang et al. (1992) Histochem. J. 24:102; Leventhal et al. (1993) Transplantation 55:857); hyperacute allograft and hyperacute xenograft rejection (Adachi et al. (1987) Trans. Proc. 19(1):1145; Knechtle et al. (1985) J. Heart Transplant 4(5):541; Guttman (1974) Transplantation 17:383); immune-complex-induced vasculitis (Cochrane (1984) Springer Seminar Immunopathol. 7:263); and myasthenia gravis (Nakano & Engel (1993) Neurology 43:1167; Barohn & Brey (1993) Clin. Neurol. Neurosurg. 95:285; Biesecker & Gomez (1989) J. Immunol. 142:2654; Lennon et al. (1978) J. Exp. Med. 147:973).
In addition, complement plays a role in ischemia reperfusion settings such as: myocardial infarction (Kilgore et al. (1994) Cardiovasc. Res. 28:437), stroke (Vasthare et al. (1993) FASEB J. 7:A424), atherosclerosis/vasculitis (Niculescu & Rus (2004) Immuno. Res. 30:73-80), renal ischemia/reperfusion (Zhou et al. (2000) J. Clinical Investigation 105:1363-1371; Arumugam et al. (2003) Kidney Intl. 63:134-142), and injury due to cardiopulmonary bypass surgery and post pump syndrome in cardiopulmonary bypass (Salama et al. (1988) N. Engl. J. Med. 318:408-14; Chenoweth et al. (1986) Complement 3:152-165; Chenoweth et al. (1981) Complement. Inflamm. 3:152-165).
Complement has also been shown to play a role in central nervous system diseases such as: multiple sclerosis (Williams et al. (1994) Clin. Neurosci. 2:229), Alzheimer's disease (Bradt et al. (1998) J. Exp. Med. 188:431; Yasoshima et al. (1999) Am. J. Pathol. 154:927), and experimental allergic neuritis (Vriesendorp et al. (1995) J. Neuroimmunol. 58:157; Feasby et al. (1987) Brain Res. 419:97).
Various other diseases, disorders, or injury that complement has been linked to include but are not limited to: fatal complication in sepsis (Hack et al. (1989) Am. J. Med. 86:20-26); hemolytic anemia (Schreiber & Frank (1972) J. Clin. Invest. 51:575); adult respiratory distress syndrome (Zilow et al. (1990) Clin. Exp. Immunol. 79:151-57; Langlois et al. (1989) Heart Lung 18:71-84; Robbins et al. (1987) Am. Rev. Respir. Dis. 135:651); thermal injury (burn, frostbite) (Gallinaro et al. (1992) Surg. Gynecol. Obstet. 174:435; Gelfand et al. (1982) J. Clin. Invest. 70:1170; Demling et al. (1989) Surgery 106:52-9); extracorporeal dialysis and blood oxygenation (Pekna et al. (1993) Clin. Exp. Immunol. 91:404; Deppisch et al. (1990) Kidney Inst. 37:696-706; Kojima et al. (1989) Nippon Jenzo Gakkai Shi 31:91-97); intestinal inflammation of Crohn's disease which is characterized by the lymphoid infiltration of mononuclear and polymorphonuclear leukocytes (Ahrenstedt et al. (1990) New Engl. J. Med. 322:1345-1349); and toxicity and side effects observed from recombinant IL-2 immunotherapy treatment (Thijs et al. (1990) J. Immunol. 144:2419), and complement activation known to occur in monoclonal antibody therapy.
In many of the diseases, disorders, and injuries listed above, experiments have shown that inhibition of complement activation can stop the progression of the disease, disorder, or injury and, in some cases, even reverse some of the damage already sustained. Thus, compounds that potently and selectively inhibit the complement cascade and/or its various components and factors will have therapeutic applications in several diseases, disorders, or injuries, including those listed above (Makrides (1998) Pharmacol. Rev. 50:59-87; Spiegel et al., Strategies for Inhibition of Complement Activation in the Treatment of Neurodegenerative Diseases in: Neuroinflammation: Mechanisms and Management, Wood (ed.), Humana Press, Inc., Totowa, N.J., Chapter 5, pp. 129-176; and U.S. Pat. No. 4,916,219; U.S. Pat. No. 6,319,897; U.S. Pat. No. 6,515,002; U.S. Pat. No. 6,232,296).